Dihydrovinblastine



Nov. 14, 1967 N. NEUSS ETAL 3,352,868

DIHYDROVINBLASTTNE Filed April 1, I964 NUCLEAR MAGNETIC RESONANCE SPECTRUM OF DIHYDROVINBLASTINE NORBERT NEUSS MARVIN GORMAN ATTORNEY United States Patent 3,352,868 DIHYDROVINBLASTINE Norbert Neuss and Marvin Gorman, Indianapolis, Ind., assignors to Eli Lilly and Company, Indianapolis, Ind., a corporation of Indiana Filed Apr. 1, 1964, Ser. No. 356,588 2 Claims. (Cl. 260--287) ABSTRACT OF THE DISCLOSURE The preparation and structure of dihydrovinblastine, effective in prolonging the life of mice infected with leukemic cells, is set forth.

COOCH wherein R is methyl, formyl, or hydrogen, respectively. In the above formula when R is methyl, the compound is denominated vinblastine; when R is formyl, the compound is denominated vincristine; and when R is hydrogen, the compound is denominated desmethylvinblastine. Two of the compounds provided by this invention, dihydrovinblastine and dihydrovincristine, are useful in inhibiting the growth of transplanted tumors in mice, particularly P-l534 leukemia. Dihydrovinblastine is particularly outstanding in this regard. For example, a dose of 20 mg. of dihydrovinblastine per mouse on days 1, 6, and 10, giving a total dose of 60 mg., results in a 173 percent prolongation of life in the mice, a result strictly comparable with that obtained by giving 1.5 mg. of vinblastine itself on days 1, 6, and 10 with a total dose of 4.5 mg. per mouse. Quite unexpectedly, however, the toxicity of dihydrovinblastine does not parallel its antitumor properties. For example, mice readily tolerate a single dose of 9-0 mg./ kg. of dihydrovinblastine, whereas a single dose of mg./ kg. of vinblastine per mouse causes a high degree of mortality. Although no data is available with regard to the use of either dihydrovinblastine or dihydrovincristine in the treatment of malignancies in humans it will be readily apparent that the fact that these compounds do inhibit the growth of a transplanted leukemia in mice is extremely interesting in itself, in that it is only by a study of the minimal structural requirements for the inhibition of transplanted tumors in mice among the Vinca alkaloids that those truly skilled in the art will be able to prepare more active and less toxic compounds or will be able to illuminate the mechanism of action of this group of compounds against tumor cells.

Dihydrodesmethylvinblastine is a useful intermediate for the preparation of dihydrovincristine. The latter compound is prepared from the former by means of a fonnylation procedure, as set forth in the copending application of Marvin German, Ser. No. 233,917, filed Oct.

29, 1962, now abandoned.

The free bases provided by this invention are amorphous, low-melting solids, which are readily converted to white, crystalline acid addition salts when contacted with suitable acids. These acid addition salts are in general high-melting solids. Among the acids which can form salts with the amine bases of this invention are the following: hydrochloric acid, sulfuric acid, 'hydrobromic acid, maleic acid, tartaric acid, malic acid, phosphoric acid, succinic acid, 2,4-dinitrobenzoic acid, and the like.

The dihydro compounds of this invention can be differentiated from their parent compounds by means of both thin layer chromatography and nuclear magnetic resonance spectral data. FIGURE 1 gives the nuclear magnetic resonance spectrum of dihydrovinblastine which has several points of difference from the nuclear magnetic spectrum of vinblastine. Differentiation of a dihydro compound from the parent compound by means of thin layer chromatography is readily illustrated by reference to the pair, vinblastine and dihydrovinblastine, using silica as the adsorbent and 100 percent methanol as the solvent. It has been found, under these experimental conditions, that vinblastine has an R, value of 0.43 whereas dihydrovinblastine is somewhat less polar and has a slightly larger R, value of 0.48. In this thin layer chromatography procedure, the substances are spotted on the chromatography plate in the form of an acid addition salt. A drop of hexane saturated with ammonium hydroxide is then added to each spot to liberate the free base, which then moves up the surface of the plate in the presence of the methanol Five grams of vinblastine sulfate and 3 ml. of 12 N hydrochloric acid were added to 200 ml. of percent ethanol. The mixture was placed in a low-pressure hydrogenation apparatus and hydrogenated at a hydrogen pressure of 30 p.s.i. for 1.25 hours at room temperature using 2 g. of palladium oxide as a catalyst. The hydrogenation mixture was filtered to remove the catalyst. The filtrate was concentrated by evaporation in vacuo and then cooled. Dihydrovinblastine dihydrochloride crystallized from the reaction mixture upon standing. After recrystallization from a mixture of methylene dichloride, ethanol, and ether, the compound melted with decomposition in the range 236-242 C.; [a] =32 (water; c.=1.0).

Dihydrovinblastine dihydrochloride was converted to the corresponding sulfate by dissolving the hydrochloride salt in ethanol and adding 1% ethanolic sulfuric acid. Dihydrovinblastine sulfate crystallized from the reaction mixture after concentration and cooling. The sulfate salt melted with decomposition at about 255 C. after recrystallization from ethanol; [oc] =+29.2 (water;

Other acid addition salts are prepared by converting dihydrovinblastine dihydrochloride or sulfate to the free base and then contacting a solution of the free base, preferably in ether, with an equivalent amount of the desired acid, also preferably in ether. The resulting acid addition salt of dihydrovinblastine can be isolated either as an in- 12 M hydrochloric acid were added, and the mixture was hydrogenated in a low-pressure, hydrogenation apparatus as described in Example 1. After the theoretical quantity of hydrogen had been adsorbed, the catalyst was separated by filtration and the filter cake washed thoroughly with a mixture of methanol and glacial acetic acid. The resulting filtrate and washings were combined and concentrated by evaporation in vacuo. The concentrate was diluted with water and the mixture made alkaline by the addition of 10 percent aqueoussodium hydroxide. Di-

hydrovinblastine free base, being insoluble in the alkaline layer, separated and was extracted into methylene. dichloride. The methylene dichloride layer was separated and 'dried and the methylene dichloride evaporated therefrom by evaporation in vacuo. The residue comprising the dihydrovinblastine was crystallized from ether and melted at about l98-202 C.

Example 3 Five grams of vinblastine sulfate were dissolved in '50 ml. of water. One hundred and fifty milliliters of ethanol containing 3 ml. of 12 N hydrochloric acid were added, followed by 3 g. of platinum oxide, and the mixture was placed in a low-temperature hydrogenation apparatus and shaken in an atmosphere of hydrogen for 1.25 hours. The catalyst was then removed by filtration and the filtrate was concentrated in vacuo to a residual volume of about 50 ml. Ether was added to the concentrate to the point of turbidity and the resulting solution was seeded 4 with authentic 'dihydrovinblastin'e dihy'drochloride crystals and cooled. A first crop of 3.2 g. of dihydrovinblastine dihydrochloride was obtained.

Di-hydrovincristine is prepared by hydrogenating desmethylvinblastine in accordance with the above procedure and then formylating dihydrodesmethylvinblastine in accordance with the procedure set forth in the copending application of Marvin Gorman, Ser. No. 233,917, filed Oct. 29, 1962,now abandoned, for producing vincristine from desmethyl'vinblas't'ine which procedure involves refiuxing a vincristine salt such as vincristine sulfate at a pH of about 1 to about 3 for from 10 to 20 hours.

We claim:

1. Dihydrovinblastine having the formula 2. An acid addition salt of a compound according to claim 1.

No references cited.

r WALTER 'A. MODANCE, Primary Examiner.

JAMES A. PATTEN, Assistant Examiner. 

1. DIHYDROVINBLASTINE HAVING THE FORMULA 